PT1409467E - A n-acylsphingosine glucosyltransferase inhibitor - Google Patents

Abstract

Disclosed is a novel enantiomeric synthesis of ceramide-like inhibitors of UDP-glucose: N -acylsphingosine glucosyltransferase. Also disclosed are novel intermediates formed during the synthesis.

Description

ΕΡ 1 409 467 / ΡΤ

DESCRIPTION " N-acyl-sphingosine-glucosyltransferase inhibitor "

BACKGROUND OF THE INVENTION

Glycosphingolipids (GSLs) are a class of naturally occurring compounds that have a multitude of biological functions, including the ability to promote cell growth, cell differentiation, adhesion between cells or between cells and matrix proteins, binding of microorganisms and viruses to cells, and tumor metastasis cells. GSLs are derived from glucosylceramide (GlcCer), which is produced from ceramide and UDP-glucose by the enzyme UDP-glucose: IV-acyl-sphingosine-glucosyltransferase (GlcCer-synthase). The structure of ceramide is shown below:

O

OH

Ceramide Accumulation of GSLs has been linked to a number of diseases, including Tay-Sachs, Gaucher and Fabry diseases (see, for example, U.S. Patent No. 6051598). GSLs have also been linked to certain cancers. For example, it has been found that certain GSLs occur only in tumors or at abnormally high concentrations in tumors; exert marked stimulatory or inhibitory actions on tumor growth when added to tumor cells in culture media; and inhibit the body's normal immune system when extravasated by tumors into the surrounding extracellular fluid. The composition of a tumor GSL is modified as the tumors become increasingly malignant and antibodies to certain GSLs inhibit tumor growth. 2 ΕΡ 1 409 467 / ΡΤ

Compounds which inhibit GlcCer-synthase may reduce GSL concentrations and have been reported to be useful for the treatment of a subject with one of the above-mentioned diseases. A number of potent GlcCer inhibitors, referred to herein as " ceramide-like amino compounds ", are disclosed in U.S. Patents Nos. 6051598, 5952370, 5945442, 5916911 and 6030995. The term " ceramide compounds " refers to ceramide analogues wherein: 1) the primary alcohol is replaced by a substituted amino group; and 2) the alkenyl group is substituted by an aryl group, preferably phenyl or substituted phenyl. The corresponding N-deacylated compounds are referred to as " sphingosine type compounds ".

Unfortunately, known processes for preparing amino compounds of the ceramide type are poorly suited for industrial scale manufacture. Due to the two chiral centers, most of the known syntheses generate four diastereoisomers, which results in the need to separate the diastereomers by chromatography and to isolate the desired enantiomer by crystallization after derivatization with optically active reagents, eg, dibenzoyl tartaric acid isomers ( see, for example, Inokuchi and Radin, Journal of Lipid Research 28: 565 (1987)). None of the processes is amenable to large-scale preparation. The enantioselective synthesis of ceramide-type amino compounds using diastereoselective reductions has been reported (Mitchell, et al., J. Org. Chem. 63: 8837 (1998) and Nishida, et al., SYNLETT 1998: 389 (1998)), but requires more than ten steps, some of which use expensive reagents such as diisobutylaluminum hydride (DIABAL) and Garner's tert-butyl ((R) - (+) - 4-formyl-2,2-dimethyl-3-oxazolidinecarboxylate ). Thus, there is a critical need for an enantioselective synthesis of ceramide-like amino compounds which is more economical and efficient, and involves fewer steps than the known syntheses.

Lee et al. (J. Biol. Chem., 247 (21), pp. 14662-14669 (1999)) disclose specific glucosylceramide synthase inhibitors. In particular, Lee et al. disclose only compounds having a C16 alkanoyl (palmitoyl) group on the nitrogen atom of the amide. 3 ΕΡ 1 409 467 / ΡΤ

SUMMARY OF THE INVENTION Here is provided an efficient and highly enantioselective synthesis of ceramide-like amino compounds. This synthesis of ceramide-like amino compounds involves only five steps from known compounds. For example, the comparative ceramide compound designated " Compound 5 " in Figure 1 was produced in an enantiomeric excess of at least 99.6% and in an overall yield of 9% (see Comparative Example 1 and 2). New intermediates prepared in the course of the synthesis are also disclosed.

In a first aspect, the invention relates to a compound according to claim 1. A further embodiment of the compound is set forth in claim 2. The invention in other aspects provides the uses of the compounds as set forth in claims 3 to 9.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic showing the synthesis of comparative Ceramide Compound (5) using the process disclosed herein. Figure 2 is a schematic showing the synthesis of comparative Ceramide Compound (13) using the procedures disclosed herein. Figure 3 shows the structures of Compounds (5) - (8).

Detailed Description of the Invention

Examples of the specific conditions for carrying out the reactions described herein are provided in Comparative Examples 1 and 2. The term " enantiomer " as used herein, and structural formulas illustrating an enantiomer are intended to include the " pure " enantiomer, free from its isomer, as well as mixtures of the enantiomer and its optical isomer wherein the enantiomer is present in an enantiomeric excess, eg, at least 10%, 25%, 50%, 75%, 90%, 95% %, 98%, or 99% enantiomeric excess.

Compounds of this invention having a sufficiently acidic, sufficiently basic, or both functional groups may accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Physiologically acceptable salts are provided. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, benzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gammahydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate and mandelate.

Base addition salts include those derived from inorganic bases, such as ammonium, alkali or alkaline earth metal hydroxides, carbonates and bicarbonates. Such bases useful in the preparation of the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide and potassium carbonate. 5 ΕΡ 1 409 467 / ΡΤ

Exemplification

Comparative Example 1 - Small scale preparation of ceramide compounds

Intermediate 1 (1R, 35,55,8a, 5) -1,3-Bis- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -5-phenyl-tetrahydro oxazolo [4,3-c] [1,4] oxazin-8-one

To a stirred solution of (5S) -5-phenylmorpholin-2-one (2.00 g, 11.3 mmol) (prepared as in Dellaria, JF Santarsiero, BD, J. Org. Chem., 1989, 54, 3916) and 1,4-benzodioxan-6-carboxaldehyde (5.56 g, 33.9 mmol) in toluene (125 mL) was added 4A molecular sieves (approximately 20 mL). The resulting amber gum was flash chromatographed on silica (diethyl ether / hexane) to provide a pale yellow solid This material was further purified by trituration with diethyl ether to give 1.89 g (34%) of product as a fluffy white solid: ¹H NMR (CDCl3) δ 7.31-7.17 (m, 5H), 6.95-6.79 (m, 5H), 5.32-5.30 (M, 2H), 4.43-4.28 (m, 2H), 4.24 (s, 4H), 4.18 (m, 4H), 4.16-4.08 (m, 2H) ppm.

Intermediate 2 (25, 3R, 1α) -3- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " phenyl) -ethylamino) -1-pyrrolidin-1-yl-propan-1-one

To a stirred solution of Intermediate 1 (1.80 g, 3.69 mmol) in chloroform (20 mL) was added pyrrolidine (2.0 mL, 24 mmol). The solution was stirred overnight and then concentrated. The resulting adhering colorless foam was taken up in methanol (16 mL) and 1N hydrochloric acid (4 mL). The mixture was refluxed for 1 hour, treated with 1N hydrochloric acid (2 mL) and refluxed for another 2 hours. The reaction solution was concentrated and the resulting residue partitioned between ethyl acetate and an aqueous solution of sodium bicarbonate. The organic layer was dried (sodium sulfate) and concentrated. The resulting pale yellow gum was purified by flash chromatography on silica gel (dichloromethane / 2N methanolic ammonia) to provide 1.40 g (92%) of Intermediate 2 as a colorless foamy solid: 1 H NMR (CDCl 3) δ 7.31-7 (M, 3H), 4.47 (d, J = 8.5, 1H), 4.18 (s, 4H), 3.82 (t, J = 5.9, 1H), 3.74 (d, J = 6.0, 2H), 3.06 (d, J = 8.5,1H), 3.06-2.97 (m, 1H), 2.92-2.83 (m, 1H), 1.97-1.87 (m, 1H), 1.45-1.15 (m, 4H) ppm.

Intermediate 3 (1R, 2R, 1S) -1- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2- (2'- phenylaminoethyl) -3-pyrrolidin-1-yl-propan-1-ol

To a stirred solution of Intermediate 2 (1.38 g, 3.35 mmol) in tetrahydrofuran (30 mL) was added lithium aluminum hydride (0.26 g, 6.9 mmol). The foam suspension was stirred overnight and then neutralized by the addition (dropwise until frothing) of 1N aqueous sodium hydroxide solution (13 mL). The mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried (sodium sulfate) and concentrated to provide a colorless gum. Flash chromatography on silica gel (dichloromethane / 2N methanolic ammonia) Î'1 409 467 / ΡΤ provided 0.94 g (70%) of product as a colorless, adherent foam: 1 H NMR (CDCl3) δ 7.36-7.17 (m, 5H), 6.88-6.74 (m 3H), 4.42 (d, J = 5.4, 1H), 4.26 (s, 4H), 3.79-3, (M, 1H), 3.55-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.67-2.68 (m, 57 (m, 1H), 2.43-2.32 (m, 4H), 2.25-2.15 (m, 1H), 1.75-1.65 (m, 4H) ppm.

Intermediate 4 (1R, 2R) -2-Amino-1- {2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -3-pyrrolidin-1-yl-propan-1 -ole

Into a high pressure reaction pump equipped with a mechanical stirrer was charged a solution of Intermediate 3 (0.91 g, 2.28 mmol) in 10: 1 methanol / water (22 mL), trifluoroacetic acid (0.18 mL, 2.3 mmol) and 20% palladium hydroxide on carbon (Perlman's catalyst: 0.91 g). The reactor was evacuated and filled with argon three times and then evacuated and filled with hydrogen (100 psi). The reaction mixture was stirred for 2 days and then evacuated and blown with nitrogen. The reaction solution was filtered through Celite and concentrated. The resulting gray-green gum was flash chromatographed on silica gel (dichloromethane / 2N methanolic ammonia) to afford 0.165 g (26%) of product as an almost colorless gum: 1 H NMR (CDCl 3) δ 6.96-6.76 (m, 3H), 4.54 (d, J = 3.7, 1H), 4.25 (s, 4H), 3.43 (s, 1H), 3.14-3.07 (m, 1H ), 2.68-2.41 (m, 6H), 1.82-1.71 (m, 4H) ppm.

Compound 5 (1R, 2R) -hexadecanoic acid [2- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethylethyl] ΕΡ 1 409 467 / ΡΤ

To a stirred solution of Intermediate 4 (0.165 g, 0.593 mmol) in dichloromethane (8 mL) was added palmitoyl chloride (0.18 g, 0.59 mmol) followed by N, N-diisopropylethylamine (0.11 mL, 0.65 mmol). The solution was stirred for 2 hours and then concentrated. The residue was partitioned between ethyl acetate and an aqueous solution of sodium bicarbonate. The organic layer was dried (sodium sulfate) and concentrated. The resulting off-white solid was flash chromatographed on silica gel (dichloromethane / 2N methanolic ammonia) to provide 0.174 g (57%) of product as a white solid. Comparisons by 1H NMR spectroscopy and analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm; eluent: 0.5% trifluoroacetic acid in hexane / dichloromethane / ethanol 67:31: 2, flow rate: 1 mL / min;

detection 280 nM) demonstrated that this material was identical to a sample of the same compound prepared by the method of Polt, et al. (J. Org. Chem., 1998, 63, 8837). The enantiomeric excess was determined to be 99.6%. The total contamination from the two possible diastereoisomers was determined to be 0.2%. Δ NMR (CDCl3) δ 6.88-6.73 (m, 3H), 5.84 (d, J = 7.3, 1H), 4.90 (d, J = 3.8, 1H), 4. (M, 1H), 2.86-2.72 (m, 2H), 2.72-2.55 (m, 4H), 2.10 (t, J = 1.5, 2H), 1.82-1.74 (m, 4H), 1.58-1.46 (m, 2H), 1.32-1.16 (m, 24H) , 0.88 (t, J = 6.7, 3H) ppm.

Comparative Example 2 - Large-scale preparation of ceramide (5S) -5-Phenylmorpholin-2-one compounds

Ph.

Ph. CHjCN -► ΌΗ

Br H 1

9 Ε 1 409 467 / ΡΤ

A solution of S - (+) - phenylglycinol (Aldrich, 10.17 g, 78.12 mmol) and diisopropylethylamine (Aldrich, 34 mL, 195 mmol, 2.5 equivalents) in CH3 CN (200 mL) was prepared. This solution was added to phenyl α-bromoacetate (18.48 g, 85.9 mmol, 1.1 equivalents) dissolved in CH 3 CN (50 mL) under nitrogen, dropwise over 2 hours. The resulting solution was stirred under nitrogen for 16-20 hours. The solvent was removed by rotoevaporation, keeping the bath temperature below 25øC. To the oil was added ethyl acetate (120 mL) and the mixture was stirred for 15 minutes. The resulting white precipitate was removed by filtration and the solid washed with ethyl acetate (25 mL). The filtrate was rotoevaporated to an oil, keeping the bath temperature below 25 ° C. After drying under vacuum for 0.5 hours, the oil was dissolved in CH 2 Cl 2 (17 mL) and loaded onto a silica gel column (60 g filled with 10% ethyl acetate / hexanes). The upper byproduct stains were eluted with 10% ethyl acetate / hexanes and the product eluted with 50% ethyl acetate / hexanes-100% ethyl acetate. The fractions containing the product were rotoevaporated to an oil, keeping the bath temperature below 25 ° C. This oil was dissolved in ethyl acetate (12 mL) and hexanes (60 mL) were added slowly in an ice bath to precipitate the product. The resulting precipitate was filtered. The white to yellow solid was dried under vacuum. The (5S) -5-phenyl-morpholin-2-one obtained (7.4 g, 41.8 mmol, 53%) was used directly in the next step.

Intermediate 1 (1R, 35,55,8a) -1,3-Bis- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -5-phenyltetrahydro-oxazole [4,3-c] [1,4] oxazin-8-one

(5 S) -5-Phenylmorpholin-2-one (7.4 g, 41.8 mmol) and benzodioxolane-6-carboxaldehyde (Aldrich or Alfa 10 ΕΡ 1 409 467 / Diss

Aesar, 20.56 g, 125.2 mmol, 3.0 equivalents) in toluene (180 mL). The solution was placed in a soxhlet extractor apparatus filled with 4Å molecular sieves (about 30 g). The solution was refluxed under nitrogen for 2-3 days. After cooling to room temperature, the solvent was removed by rotoevaporation and the oil was dissolved in ethyl acetate (200 mL). A solution of sodium bisulfite (Aldrich, 50 g) in water (100 ml) was added and the two phase mixture was stirred at room temperature for 1 hour. The resulting white solid was removed by filtration and washed with ethyl acetate. The filtrate was placed on a separatory funnel and the layers separated. The organic layer was washed with water (100 mL) and brine (100 mL). The dried solution (Na 2 SO 4) was filtered and roto-evaporated to a yellow-red foamy oil (23.11 g). After drying under vacuum for 1 hour, diethyl ether (350 ml) was added and the mixture was stirred at room temperature for 16-20 hours. The resulting white-yellow solid was filtered. The solid was dried under vacuum. The cycloadduct was obtained in 46% yield (9.34 g).

Intermediate 2 (2 S, 5 R, 1 R) -3- (2 ', 3'-Dihydrobenzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " hydroxy-1'-phenyl-ethylamino) -1-pyrrolidin-1-yl-propan-1-one

To the cycloadduct (Intermediate 1, 6.7 g, 13.74 mmol) dissolved in dichloromethane (40 mL) was added pyrrolidine (Aldrich, 5.7 mL, 68.7 mmol, 5 equivalents). The solution was stirred under nitrogen at room temperature for 16-18 hours. The solvent was rotoevaporated to provide a yellow foamy oil which was dried under vacuum for 0.5 hours. The crude product was dissolved in methanol (115 mL) and 1M aqueous HCl (115 mL) was added. The solution was refluxed for 4 hours. After cooling to ambient temperature, the methanol was removed by roto-evaporation. Ethyl acetate (60 mL) was added and the two phase system was stirred at room temperature for 5-15 minutes. The two layers were separated and the organic layer was extracted with 1 M HCl (30 mL). The combined aqueous layers were washed with ethyl acetate (60, 30 mL). Saturated sodium bicarbonate solution (150 mL) was slowly added to the aqueous layer. The product was extracted three times with ethyl acetate (60 mL) from the basic aqueous layer (pH = 8-9). The combined organic layers containing the product were washed with brine (30 mL). After drying with Na 2 SO 4 the solution was filtered and roto-evaporated to give a yellow solid. Intermediate 2 was obtained in 93% yield (5.26 g).

Intermediate 3 (1α, 2R, 1α) -11- (2 ', 3'-Dihydrobenzo [1,4] dioxin-6'-yl) -2- (2-hydroxy-1'- ethylamino) -3-pyrrolidin-1-yl-propan-1-ol

OH OH

OH To a 3-necked flask equipped with addition funnel and condenser were added LiAlH 4 (Aldrich, 1.2 g, 31.7 mmol, 2.5 equivalents) and anhydrous THF (20 mL) under nitrogen. A solution of Intermediate 2 (5.23 g, 12.68 mmol) in anhydrous THF (75 mL) was added dropwise to the reaction over 15-30 minutes. The reaction was refluxed under nitrogen for 9 hours. The reaction was cooled in an ice bath and 1M NaOH solution was carefully dropwise added. After stirring at room temperature for 15 minutes, water (50 mL) and ethyl acetate (75 mL) were added. The layers were separated and the aqueous layer extracted twice with ethyl acetate (75 mL). The combined organic layers were washed with brine (25 mL). After drying with Na 2 SO 4, the solution was filtered and roto-evaporated to provide a colorless foam oil 12 Ρ Ρ 1 409 467 / ΡΤ in yellow. Intermediate 3 was obtained in 99% yield (5.3 g).

Intermediate 4 (1R, 2R) -2-Amino-1- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -3-pyrrolidin-1-yl-propan-1-ol

Intermediate 3 (5.3 g, 13.3 mmol) was dissolved in methanol (60 mL). Water (6 mL) and trifluoroacetic acid (2.05 mL, 26.6 mmol, 2 equivalents) were added. After being placed under nitrogen, 20% palladium hydroxide on carbon (Pearlman, Lancaster or Aldrich catalyst, 5.3 g) was added. The mixture was placed in a Parr Pressurized Reactor apparatus with glass insert. The device was placed under nitrogen and then under a hydrogen pressure of 110-120 psi. The mixture was stirred for 2-3 days at room temperature under hydrogen pressure of 100-120 psi. The reaction mixture was placed under nitrogen and filtered through a pad of celite. The celite pad was washed with methanol (100 mL) and water (100 mL). The methanol was removed by roto-evaporation. The aqueous layer was washed with ethyl acetate three times (100, 50, 50 mL).

A 10M NaOH solution (10 mL) was added to the aqueous layer (pH = 12-14). The product was extracted from the aqueous layer three times with dichloromethane (100, 100, 50 mL). The combined organic layers were dried over Na2SO4, filtered and rotoevaporated to a colorless oil. The foamed oil was dried under vacuum for 2 h. Intermediate 4 was obtained in 90% yield (3.34 g).

Compound 5 (1R, 2aS, 3aS) -Dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] 2R) -hexadecanoic acid 13 ΕΡ 1 409 467 / ΡΤ

To a solution of Intermediate 4 (3.34 g, 12.0 mmol) in dichloromethane (50 mL) was added a solution of palmitic acid N-hydroxysuccinimidoester (Sigma, 4.24 g, 12.0 mmol) for 15-30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. To the reaction was added dichloromethane (50 mL) and 1M NaOH solution (25 mL). The two-phase system was stirred at room temperature for 15-30 min. Water (25 mL) was added and the layers were separated. The aqueous layer was extracted again with dichloromethane (25 mL). The combined organic layers were washed twice with water (25 mL) and once with a saturated sodium chloride solution (25 mL). The organic layer was dried over Na2SO4, filtered and rotoevaporated to a pale yellow oil. The crude product was recrystallized from hexane (50 mL). The white solid (5.46 g) obtained was separated on silica gel (300 g) with 2% methanol: 4% dichloromethane-methanol: 2M dichloromethane-ammonium in 4% methanol: dichloromethane. The white solid obtained was recrystallized from hexanes (70 mL). Compound 5 was obtained in 66% yield (4.18 g). Analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm; eluent: 0.5% trifluoroacetic acid in 67: 31: 2 hexane / dichloromethane / ethanol: 1 mL / min; detection: 280 nM) showed that this material was 98.98% pure with 0.89% of a diastereoisomer and 0.14% of the enantiomer.

Comparative Example 3 - Large-scale alternative preparation of ceramide-type compounds

(5 S) -5-Phenylmorpholin-2-one HCl salt

A solution of phenyl bromoacetate (Aldrich, 862.17 g, 4.0 mol, 1.1 equivalents) in acetonitrile (reagent quality, 1500 ml) was cooled in an ice bath (internal temperature less than 5øC). To this was added a cold slurry (internal temperature less than 5 ° C) of S - (+) - 2-phenylglycinol (Aldrich, 500 g, 3.65 moles, 1 equivalent) and diisopropylethylamine (DIPEA) (Aldrich, 1587 ml, 9.11 moles, 2.5 equivalents) in acetonitrile (2900 ml) was added portionwise while the internal temperature was maintained below 10 ° C. The mixture was stirred at this temperature for 30 minutes before the ice bath was removed and the mixture was left under stirring at room temperature additionally for 4 hours. The solvent was removed under vacuum while maintaining the bath temperature at 25 ° C. The mixture was coevaporated with ethyl acetate (2 x 500 mL) to give a pale yellow viscous oil. To this reaction mixture was added ethyl acetate (4500 mL) and the flask was immersed in an ice bath with stirring. The mixture was allowed to cool below 8 ° C. The solid was filtered and washed with ethyl acetate (3x250 mL). The solution was cooled to below 5 ° C. Dry HCI was slowly passed through the solution while maintaining the internal temperature below 15øC until the pH was less than 2 (wet pH paper). The mixture was allowed to stir at this temperature and this pH for an additional 20 minutes before the solid was filtered by suction. The solid was washed with ethyl acetate (3 x 200 mL) and dried under high vacuum for about 20 hours. The yield was 412 g (53%). The NMR spectrum was consistent with the (5S) -5-phenylmorpholin-2-one HCl salt.

Intermediate 1 (1R, 35,5S, 8aS) -1,3-Bis- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -5-phenyltetrahydro oxazolo [4,3-c] [1,4] oxazin-8-one To a stirred suspension of (5S) -5-phenyl-morpholin-2-one HCl salt (381 g, 1 equiv.) in acetate 15% ethyl acetate in toluene (2270 ml) was added a solution of sodium bicarbonate (1.1 equivalents) in water (2000 ml). The resulting biphasic solution was stirred at room temperature for about 1 hour. The organic layer was transferred to a flask containing 1,4-benzodioxan-6-carboxaldehyde. The flask was then fitted with a Dean-Stark unit, a condenser and a nitrogen inlet. The mixture was heated to reflux with stirring while about 650 ml of solvent (mixture of ethyl acetate and toluene) were collected via Dean-Stark unit. The resulting red-yellow solution was allowed to reflux under nitrogen for about 64 hours while the water formed during the reaction was collected in the Dean-Stark unit. Most of the solvent was then removed by distillation at atmospheric pressure through the Dean-Stark unit. The residual solvent was then removed by coevaporation with heptane (500 mL) and tert-butyl methyl ether (2 x 725 mL) to afford a yellow semi-solid. The semi-solid product was dissolved in ethyl acetate (3400 ml). A solution of sodium bisulfite (920 g) in water (1500 ml) was added and the mixture was allowed to stir at room temperature for about 1 hour. The solid which formed was removed by filtration and washed with ethyl acetate (3.times.400 ml). The filtrate was washed with water (1450 ml), 5% brine solution (1450 ml) and dried over MgSO4 (100 g). The solvent was removed in vacuo to provide a yellow solid. To this was added tert-butyl methyl ether (2900 ml) and the suspension was stirred at room temperature for 20 to 22 hours. The yellow solid was suction filtered, washed with tert-butyl methyl ether (2 x 600 mL) and dried under high vacuum at room temperature for about 22 hours. The yield was 400.5 g (58%). The ¹H NMR and TLC values were consistent with Intermediate 1.

Intermediate 2 (25,3-R, 1c) -3- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -3-hydroxy-2- (2 " phenyl) -ethylamino) -1-pyrrolidin-1-yl-propan-1-one

A solution of Intermediate 1 (312 g, 0.64 moles), pyrrolidine (267 ml, 3.2 mol, 5 equivalents) and tetrahydrofuran (1350 ml) was heated at reflux for 4.5 hours under a nitrogen atmosphere. The solvent and excess pyrrolidine were removed in vacuo to give the crude Intermediate as an orange viscous oil. The oil was dissolved in methanol (3000 ml) and 1M hydrochloric acid solution (3000 ml). The resulting solution was heated at reflux for about 7 hours. The solvent was then removed under vacuum to provide a mixture of an oil and water. To this was added ethyl acetate (2000 ml) and the aqueous layer was separated. The organic layer was extracted with 1M aqueous HCl (1000 ml). The aqueous layers were combined and washed with ethyl acetate (2000 ml). The aqueous layer was cooled in a bath of 16 æl 1 409 467 / æ ice. Ο ρΗ of the aqueous layer was adjusted to about 9 (pH paper) with 10M aqueous NaOH (525 ml). The aqueous layer was extracted with ethyl acetate (3000 ml). The organic layer was washed with a solution of 5% brine (1000 ml) and dried (Na2 SO4). The solvent was removed in vacuo to give a yellow viscous oil. The yield was 213.4 g, 81%. The ¹H NMR spectrum was consistent with Intermediate 2.

Intermediate 3 IR, 2R, 1 " 5) -1- (2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2- (2'-hydroxy-1'-phenyl -ethylamino) -3-pyrrolidin-1-yl-propan-1-ol

To a slurry of LiAlH 4 (50.7 g, 1.34 moles, 2.6 equivalents) in tetrahydrofuran (700 ml) was added a solution of Intermediate 2 (213.34 g, 0.517 mol) in tetrahydrofuran (2000 ml) slowly with stirring at room temperature. The mixture was refluxed for about 4 hours. TLC analysis (10% methanol in dichloromethane, v / v) indicated consumption of the starting material. The reaction mixture was cooled in an ice bath (below 5øC) and water (135 ml) was added very slowly, while maintaining the internal temperature less than or equal to 10øC. To this was then added a 15% aqueous NaOH solution (70 ml) followed by water (200 ml). The reaction mixture was allowed to warm to room temperature while stirring was maintained. Dichloromethane (1000 ml) was then added to the mixture and the salts were filtered through a pad of celite. The salts were washed with dichloromethane (2x500 ml). The filtrates were combined and the solvent was removed in vacuo to give a yellow oil. The oil was dissolved in 1M aqueous HCl (1500 ml) and washed with ethyl acetate (3 x 500 ml). The aqueous layer was cooled in an ice bath to below 5øC and the pH of the aqueous layer was adjusted to 12 to 13 with a 10M aqueous NaOH solution (220 ml) keeping the internal temperature less than or equal to 10øC. The mixture was allowed to warm to room temperature. The aqueous layer was extracted with dichloromethane (2x500 ml). The organic layers were combined and washed with a solution of brine (500 mL), dried (Na2 SO4) and the solvent removed in vacuo to provide a yellow viscous oil. The yield was 186.4 g (88.5%). The 2 H NMR spectrum was consistent with Intermediate 3. 17 Î »1 409 467 / ΡΤ

Intermediate 4, dioxalate salt (1R, 2R) -2-Amino-1- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -3-pyrrolidin- propan-1-ol, dioxalate salt

A suspension of Intermediate 3 (358 g, 0.90 mol), ethanol (1500 ml), 1 M HCl solution (1500 ml) and 10% Pd (OH) 2 (32 g, 20% by weight) was hydrogenated at about 50 psi for about 36 hours at room temperature. The mixture was filtered through a Cuono filter. The Cuono filter was washed with 10% ethanol in water (500 ml). The filtrates were combined and the ethanol was removed in vacuo. The aqueous layer was extracted with ethyl acetate (3x600 ml). The organic layer was extracted with 1M aqueous HCl (700 mL). The aqueous layers were combined and cooled in an ice bath (0 to about 5 ° C). The pH of the aqueous layer was adjusted to about 12 (pH paper) with a 10M aqueous NaOH solution (490 ml) keeping the internal temperature below 10Â ° C. The aqueous layer was allowed to warm to room temperature. The aqueous layer was extracted with dichloromethane (2x1500 ml, 1x750 ml). The combined organic layers were dried over MgSO4 and the solvent was removed in vacuo to provide a viscous yellow oil. The crude weight was 214.3 g (86%). The ¹-NMR spectrum was consistent with Intermediate 4.

A solution of oxalic acid (152.4 g, 1.693 moles, 2.2 equivalents) in methylisobutylketone (2300 ml) was added slowly with stirring to a solution of Intermediate 4 (214.3 g, 0.77 mol, 1 equivalent) in methylisobutylketone (800 ml) at room temperature. The resulting mixture was stirred at room temperature for about 2.5 hours. The solid was filtered and triturated with acetone (2000 ml) at ambient temperature for about 16 hours. The solid was filtered, washed with acetone (3 x 100 mL) and dried under high vacuum to afford an off-white solid. The yield was 312.5 g (89%). The NMR spectrum was consistent with the dioxalate salt of Intermediate 4.

Compound 5 (1R, 2R) -2-Amino-2- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl- -hexadecanoic acid To a cold (about 5 ° C) solution of Intermediate 4 dioxalate salt (507 g, 1.11 mol) in water (10 L) was added a 10M aqueous NaOH solution (500 ml) with stirring, the internal temperature being maintained below 10 ° C. The solution was allowed to warm to room temperature, while the pH of the solution was maintained at about 14 (pH paper). The aqueous layer was extracted with dichloromethane (3 x 6000 ml). The organic layers were combined, washed with water (2000 ml), dried (MgSO4) and the solvent removed under vacuum to provide a yellow viscous oil, Intermediate 4. The yield was 302 g (98%). The ¹-NMR spectrum was consistent with Intermediate 4.

A solution of the NHS-ester of palmitic acid (Sigma, 382.5 g, 1.01 equiv) in dichloromethane (2500 ml) was added to a solution of Intermediate 4 (302 g) in dichloromethane (1500 ml) at room temperature for a period of 1.25 hours under a nitrogen atmosphere. The mixture was allowed to stir at room temperature for about 18 hours. Aqueous 1M NaOH solution (2425 ml) was added and the mixture was stirred at room temperature for about 3 hours. The organic layer was separated and the aqueous layer was extracted with dichloromethane (800 ml). The organic layers were combined, washed with 1M NaOH solution (3x1500 ml) and water (1500 ml). The organic layer was dried over MgSO4 and the solvent was removed in vacuo to afford a semi-solid product. The semi-solid was coevaporated with heptane (3x100 ml). The crude product was transferred to a 12 L three-necked RB flask and heptane (7500 ml) was added. The mixture was heated to reflux with stirring under a nitrogen atmosphere. The solution was slowly cooled to about 55øC (internal temperature) and poured into another vial. The solution was stirred at room temperature for 24 hours under a nitrogen atmosphere. The off-white solid was filtered, washed with heptane (2 x 500 ml) and dried under high vacuum for 24 hours. The solid (397 g) was transferred to a 12 L RB flask and 30% ethyl acetate in heptane (8000 ml) was added. The resulting mixture was heated at reflux for 30 minutes with stirring. The solution was cooled to about 55øC (internal temperature) and poured into another vial. Stirring was maintained at room temperature under a nitrogen atmosphere for about 24 hours. The solid was filtered, washed with heptane (2 x 100 mL) and dried under high vacuum to provide an off-white solid. The ε 409 467 / ΡΤ yield was 324 g (58%). NMR and TLC analyzes were consistent with Compound 5. M.p. 96.1 ° C. HPLC analysis: chiral purity 99.7%, chemical purity 99.7%. Anal. calc. for C 31 H 52 N 2 O 4: C, 72.05; H, 10.14; N, 5.42; Found. C, 72.03; H, 10.19; N, 5.42.

Example 4 - Preparation of Compounds 6-8

Esters of fatty acid IV-hydroxy succinimide were prepared by the procedure of Lapidot, Y., Rappoport, S. and Wolman, Y., Journal of Lipid Research 8, 1967, or as follows: (Aldrich, 20.0 g, 173 mmol) and triethylamine (29 mL, 208 mmol) were dissolved in dichloromethane in an ice bath under nitrogen. Octanoyl chloride (Aldrich, 35 mL, 205 mmol) was added dropwise over 0.5 hours. The ice bath was removed and the solution with a white solid was stirred for 1 hour at room temperature. The white solid was removed by filtration and the filtrate was washed with water (100 mL) and saturated aqueous sodium bicarbonate (100 mL). The organic layer was dried over sodium sulfate, filtered and heptane (100 mL) was added. The solution was rotoevaporated to remove most of the dichloromethane and left a precipitate in colorless, white, heptane in flakes. The precipitate was filtered and washed with heptane. After drying, the octanoic acid N-hydroxysuccinimide ester was obtained in 84% yield (35.4 g). Δ NMR (CDCl3) 2.84 (br s, 4H), 2.60 (t, J = 7.48 Hz, 2H), 1.78-1.71 (m, 2H), 1.42-1, 26 (m, 8H), 0.88 (t, J = 6.7 Hz, 3H) ppm.

Compound 6 (1R, 2R) -2-Amino- (2 ', 3'-dihydrobenzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl- -octanoic acid 20 Ε 1 409 467 / ΡΤ ΟΗ

CH 2 Cl 2 ΟΗ N-hydroxy succinimido ester of octanoic acid >

(CH 2) s CH 3 Compound 6

To Intermediate 4 (22.36 g, 80.33 mmol) dissolved in anhydrous dichloromethane (300 mL) was added a solution of octanoic acid IV-hydroxy succinimide ester (19.4 g, 80.39 mmol) dissolved in anhydrous dichloromethane (150 mL) for 15-30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. To the reaction was added 1M aqueous NaOH solution (200 mL). The two-phase system was stirred at room temperature for 45 minutes. The layers were separated and the combined organic layers were washed twice with 1M NaOH (2x200 mL) and twice with water (2x100 mL). The organic layer was dried over sodium sulfate, filtered and rotoevaporated to a yellow oil. Most of the crude material was dissolved in 5% ethyl acetate in refluxing heptane (1 L). After cooling to 40øC, the cloudy solution was separated from the yellow oil by decantation of the solution into a new vial. The first flask was rinsed twice with 5% ethyl acetate in heptane (2x250 mL) by the same procedure (refluxing and cooling to 40 ° C and decantation of the solution from the oil). The combined solution was heated to reflux and allowed to cool to ambient temperature over 4 hours. The resulting white solid was filtered and washed with 5% ethyl acetate in heptane (100 mL) and heptane (100 mL). The white solid (13.9 g) was dried under vacuum for 16-24 hours. This solid was mostly dissolved in 5% ethyl acetate in heptane (800 mL) at reflux. After cooling to 50øC, the cloudy solution was separated from the yellow oil by decantation of the solution into a new vial. The first flask was rinsed with 5% ethyl acetate in heptane (100 mL) by the same procedure (refluxing and cooling at 50 ° C and decantation of the solution from the oil). The combined solution was heated to reflux and allowed to cool to ambient temperature over 4 hours. The resulting white solid was filtered and washed with 5% ethyl acetate / heptane (50 mL) and 21 Å-1 409 467 / ΡΤ heptane (50 mL). After drying at room temperature under vacuum for 2-3 days, Compound 6 was obtained in 39% yield (12.57 g). The analytical HPLC was shown to be 99.9% by the analytical chiral HPLC (column: Chirex (S) -VAL and (R) -NE, 4.6x250 mm). that this material was 99.6% pure. Federal Police. 87-88Â ° C. 1 H NMR (CDCl 3) δ 6.86-6.73 (m, 3H), 5.84 (d, J = 7.3 Hz, 1H), 4.91 (d, J = 3.4 Hz, 1H) , 4.25 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.3 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 8H), 0.87 (t, J = 6.9 Hz, 3H) ppm.

Comparative compound 7 (1R, 2 ', 3'-Dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] , 2R) -nonanoic acid

N-hydroxysuccinimido ester of nonanoic acid CH 2 Cl 2

OH

Compound 7

This compound was prepared by the procedure described for Compound 6 using nonanoic acid N-hydroxysuccinimide ester. By analytical HPLC this material was shown to be 98.4% pure. Federal Police. 74-75 ° C. 1 H NMR (CDCl 3) δ 6.86-6.76 (m, 3H), 5.83 (d, J = 7.3 Hz, 1H), 4.90 (d, J = 3.3 Hz, 1H) , 4.24 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.3 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 10H), 0.87 (t, J = 6.9 Hz, 3H) ppm.

Comparative compound 8 (1R) -2- (2 ', 3'-dihydro-benzo [1,4] dioxin-6'-yl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-ethyl] , 2R) -decanoic acid

Compound 8 22 Ε 1 409 467 / ΡΤ

This compound was prepared by the procedure described for Compound 6 using decanoic acid N-hydroxysuccinimide ester. By analytical HPLC this material was shown to be 99.3% pure. Federal Police. 97.5-98.5 ° C. Δ NMR (CDCl3) δ 6.86-6.76 (m, 3H), 5.83 (d, J = 7.5 Hz, 1H), 4.90 (d, J = 3.4 Hz, 1H) , 4.24 (s, 4H), 4.24-4.18 (m, 1H), 2.85-2.75 (m, 2H), 2.69-2.62 (m, 4H), 2. , 10 (t, J = 7.5 Hz, 2H), 1.55-1.45 (m, 2H), 1.70-1.85 (m, 4H), 1.30-1.15 , 12H), 0.87 (t, J = 6.8 Hz, 3H) ppm.

Comparative Example 5 - Preparation of Compound 13

Intermediate 9 (1R, 35,55,8a, 5) -1,3-Bis- (4-benzyloxy-phenyl) -5-phenyl-tetrahydro-oxazolo [4,3- c] [1,4] oxazin- 8-one

The (5S) -5-phenylmorpholin-2-one HCl salt (57.45 g, 268.9 mmol) was stirred with ethyl acetate (500 mL) and saturated aqueous sodium bicarbonate (250 mL) for 30 minutes , until the biphasic solution becomes clear. The phases were separated, and the aqueous layer was extracted with ethyl acetate (2x250 mL). The combined organic phases were washed with brine (250 mL). The organic layer was dried over sodium sulfate, filtered, concentrated to an oil, and dried under vacuum for 60 minutes. 5- (S) -phenylmorpholin-2-one was obtained in 86% yield (40.98 g, 231.3 mmol). 5- (S) -phenylmorpholin-2-one (40.98 g, 231.3 mmol) and 4-benzyloxybenzaldehyde (Aldrich, 147.3 g, 694 mmol, 3.0 equivalents) were dissolved in toluene (750 mL ). The reaction was equipped with a Dean Stark device and a reflux condenser. The solution was refluxed under nitrogen for 2 days. After cooling to room temperature, the solvent was removed by rotoevaporation and the oil was dissolved in ethyl acetate (500 mL). A solution of sodium bisulfite (Aldrich, 125 g) was added 23 Ź 1 409 467 / ΡΤ

dissolved in water (250 mL) and the two phase mixture was stirred at room temperature for 3 hours. The resulting white solid was removed by filtration and washed with ethyl acetate. The filtrate was placed on a separatory funnel and the layers separated. The organic layer was washed with water (250 mL), brine (250 mL), and then dried (sodium sulfate), filtered and rotoevaporated to a foam oil (144 g). After drying under vacuum for 1 hour, tert-butylmethyl ether (1450 mL) was added and the mixture was stirred at room temperature for 5 hours. The resulting white-yellow solid was filtered. The solid was dried under vacuum. Intermediate 9 was obtained in 27% yield (41.64 g, 71.46 mmol). NMR (CDCl 3) δ 7.5-6.8 (m, 23H), 5.0 and 5.1 (2s, 4H), 4.5-4.3 (m, 2H), 4.2-4, 1 (m, 2H) ppm.

Intermediate 10 (2S, 3R, 1S) -3- (4-Benzyloxy-phenyl) -3-hydroxy-2- (2'-hydroxy-1'-phenyl-ethylamino) -1-pyrrolidin-1-yl -propan-1-one

OH

To Intermediate 9 (45.1 g, 77.4 mmol) dissolved in tetrahydrofuran (250 mL) was added pyrrolidine (Aldrich 33 mL, 395 mmol, 5.1 equivalents). The solution was stirred under nitrogen at room temperature for 16-18 hours. The solvent was rotoevaporated to provide a foamy yellow oil which was dried under vacuum for 0.5 hours. The crude product was dissolved in methanol (220 mL) and 1M aqueous HCl (220 mL) was added. The solution was refluxed for 4 hours. After cooling to room temperature, the methanol was removed by roto-evaporation. To the resulting oil was slowly added 10M aqueous NaOH (22 mL to adjust the pH to 14). The product was extracted three times with dichloromethane (300, 100, 100 mL) from the basic aqueous layer. After drying with sodium sulfate, the combined organic layer was filtered and roto-evaporated to provide a yellow-orange foamy solid. Tert-butyl methyl ether (300 mL) was added and the mixture was stirred at room temperature for 7 hours. The resulting white-yellow solid was filtered, washed with tert-butyl methyl ether (50 mL) and dried under vacuum. Intermediate 10 was obtained in 83% yield (29.77 g). Δ NMR (CDCl3) δ 7.4-7.2 (m, 12H), 6.9-6.8 (m, 2H), 5.05 (AB quartet, 2H), 4.47 (d, J = 8.5, 1H), 3.9-3.3 (m, 3H), 3.05 (d, J = 8.5, 1H), 3.0-2.8 (m, 2H) 3-2.2 (m, 1H), 1.85-1.7 (m, 1H), 1.45-1.15 (m, 4H) ppm.

Intermediate 11 (1R, 2R, 1S) -1- (4-Benzyloxy-phenyl) -2- (2'-hydroxy-1'-phenyl-ethylamino) -3-pyrrolidin-1-yl-propan- ol

In a 3-necked flask with addition funnel and condenser under nitrogen was added LiAlH 4 (Aldrich, 6.3 g, 166 mmol, 2.57 equivalents) and anhydrous tetrahydrofuran (75 mL). A solution of Intermediate 10 (29.7 g, 64.48 mmol) in anhydrous tetrahydrofuran (300 mL) was added dropwise to the reaction over 15-30 minutes. The reaction was refluxed under nitrogen for 9 hours. The reaction was cooled in an ice bath and water (7.0 mL) was added very carefully and dropwise (vigorous exothermic reaction, with hydrogen release). A 15% aqueous NaOH solution (7.0 mL) was added dropwise, followed by water (21 mL). At the middle of the final water addition a large amount of a white solid formed. It was stripped by the addition of dichloromethane (250 mL). After stirring at room temperature for 15 minutes, the mixture was filtered through a pad of celite (17 cm in diameter by 1 cm in height). The precipitate was washed with dichloromethane (2x250 mL). The filtrate was rotoevaporated to an oil. The oil was dissolved in 1M aqueous HCl (300 mL). This aqueous layer was washed with tert-butyl methyl ether (2x200 mL). After cooling in an ice bath, 10M aqueous NaOH (35 mL) was carefully added to the aqueous layer (final pH = 14). The product was extracted three times with 25 æl 1 409 467 / ΡΤ dichloromethane (300 mL, 200 mL and 100 mL). After drying with sodium sulfate, the solution was filtered and roto-evaporated to give a white solid. After drying, Intermediate 11 was obtained in 94% yield (26.9 g). Δ NMR (CDCl3) δ 7.46-7.115 (m, 12H), 6.98-6.96 (m, 2H), 5.08 (s, 2H), 4.49 (d, J = 4.7 , 1H), 3.70-3.65 (m, 1H), 3.60-3.55 (m, 1H), 3.54-3.45 (m, 1H), 3.00-2.90 (m, 1H), 2.7-2.6 (m, 1H), 2.36 (br s, 4H), 2.15-2.05 (m, 1H), 1.70 (br s, 4H ) ppm.

Intermediate 12 (1R, 2R) -2-Amino-1- (4-benzyloxy-phenyl) -3-pyrrolidin-1-yl-propan-1-ol,

OH-Q.H

Intermediate 11 (26.9 g, 60.24 mmol) was dissolved in methanol (400 mL) and 1M aqueous HCl (130 mL) was added. After being placed under nitrogen, 20% palladium hydroxide on carbon (Pearlman's catalyst, Aldrich, 10.8 g) was added. The reaction was placed under nitrogen and then under hydrogen by evacuation and filling of a flask. The mixture was stirred for 48 hours at room temperature under a hydrogen flask. The reaction mixture was placed under nitrogen and filtered through a pad of celite. The celite pad was washed with 10% water in methanol (250 mL) and water (50 mL). The solvent was removed by roto-evaporation and coevaporation with toluene (3x100 mL). The foamed solid was dissolved in isopropanol (300 mL) under reflux. The solution was cooled to room temperature and tert-butyl methyl ether (550 mL) was added. After stirring at room temperature for 2 hours, the white solid was filtered and washed with tert-butyl methyl ether. After drying, Intermediate 12 was obtained in about 99% yield (18 g). 1 H NMR (DMSOd 6) δ 9.68 (br s, 1H), 8.53 (br s, 2H) 7.24 (d, J = 8.55 Hz, 2H), 6.80 (d, J = 8.55 Hz, 2H), 4.72 (d, J = 7.0 Hz, 1H), 3.8-3.6 (m, 2H), 3.4-3.6 (m, 3H) , 3.0-3.2 (m, 2H), 2.7-2.5 (br s, 1H), 2.0-1.7 (br s, 4H) ppm. 26 ΕΡ 1 409 467 / ΡΤ

Compound 13 (1R, 2R) -hexadecanoic acid [2- (4-benzyloxy-phenyl) -2-hydroxy-1-pyrrolidin-1-ylmethyl-

To Intermediate 12 (16.17 g, 49.36 mmol) suspended in tetrahydrofuran (500 mL) was added triethylamine (28 mL, 4 equivalents). A solution of palmitic acid N-hydroxysuccinimidoester (Sigma, 19.2 g, 54.29 mmol) dissolved in tetrahydrofuran (125 mL) was added over 30 minutes under nitrogen at room temperature. The solution was stirred at room temperature for 18-20 hours. The white precipitate was removed by filtration and the filtrate was rotoevaporated to an off-white foamy solid (35.5 g). The crude material was dissolved in dichloromethane (500 mL) and washed with water (100 mL) and a saturated aqueous sodium carbonate solution (100 mL). After drying with sodium sulfate, the solution was filtered and roto-evaporated to give an off-white foamy solid (24, 75 g). This material was recrystallized from 40% ethyl acetate in heptane (500 mL, hot filtration). Compound 13 was obtained in 61% yield (14.45 g). Analytical chiral HPLC showed that this material was 99.7% of the desired R, R isomer. Analytical HPLC showed that this material was 99.6% pure. Federal Police. 95-97 ° C. 1H NMR (CDCl3) δ 7.15 (d, J = 8.5Hz, 2H), 6.70 (d, J = 8.5Hz, 2H), 6.0 (d, J = 7.3, 1H ), 4.96 (d, J = 3.8, 1H), 4.3-4.2 (m, 1H), 2.9-2.7 (m, 2H), 2.65-2.55 (m, 4H), 2.10 (t, J = 7.5, 2H), 1.75 (br s, 4H), 1.58-1.46 (m, 2H) 16 (m, 24H), 0.9 (t, J = 6.7, 3H) ppm.

Lisbon, 2012-07-06

Claims (9)

A compound represented by the following structural formula: ## STR2 ## (CH 2) 5 CH 3 or a physiologically acceptable salt thereof. A compound according to claim 1 in the form of the tartrate salt. A compound according to claim 1 or claim 2 for use in therapy. Use of the compound according to claim 1 or claim 2 for the manufacture of a medicament for the inhibition of GlcCer synthase or reduction of GSL concentrations in a subject in need of such treatment. Use of the compound according to claim 1 or claim 2 for the manufacture of a medicament for the treatment of Gaucher's disease. Use of the compound according to claim 1 or claim 2 for the manufacture of a medicament for the treatment of Fabry's disease. A compound according to claim 1 or claim 2 for use in inhibiting GlcCer synthase or reducing GSL concentrations in a subject in need of such treatment. A compound according to claim 1 or claim 2 for use in the treatment of Gaucher's disease. ΕΡ 1 409 467 / ΡΤ 2/2 A compound according to claim 1 or claim 2 for use in the treatment of Fabry disease. Lisbon, 2012-07-06